The invention concerns a process for the genetic transformation of cells, in particular plant cells, together with an apparatus to carry out the process for the insertion of particles in cells. Such a process and a corresponding apparatus are described for example in EP-A-0 270 356.
The present invention further concerns the application of the process according to the invention to the preparation of transgenic plants, together with transgenic plants obtainable by said process and the progeny thereof.
Numerous processes and methods are available at the present time for the genetic manipulation of the genotype of plants by means of the recombinant DNA technology; they are routinely used in many laboratories.
The most effectively investigated and most frequently used processes undoubtedly include the Agrobacterium transformation system.
Agrobacterium cells have on their Ti-plasmid a large DNA fragment, the so-called T-DNA region, which in the natural transformation of plant cells is integrated into the plant genome.
This natural gene transfer system may be used after carrying out different modifications as a gene vector system for the controlled transformation of plants (Chilton, M. D., 1983).
However, the Agrobacterium transformation system has the decisive disadvantage that the effective range of Agrobacteria is restricted to certain dicotyledonous plants and a few representative of the monocotyledons (Hernalsteens & al., 1984; Hookas-Van-Slogteren & al, 1984), which are insignificant from an agricultural economic standpoint. This signifies that the most important cultivated plants are not accessible for effective gene transfer.
Furthermore, the agrobacteria used are pathogens, which in their host plants induce characteristic disease symptoms in the form of cancer like tissue growths and which therefore may be handled under strict safety regulations in the laboratory only.
Alternative transformation systems, which were developed to equalize the disadvantages of the Agrobacterium transformation systems and which are directed at the transfer of exogenic DNA into plant protoplasts, such as the direct gene transfer of DNA in protoplasts (Paszkowski & al., 1984, Potrykus & al., 1986) and the microinjection of vector-free DNA in protoplasts (Steinbiss and Stabel, 1983; Morikawa and Yamada, 1985) or cells (Nomura and Komamime, 1986), must be considered problematic to the extent that the regeneration of entire plants from plant protoplasts of a plurality of plant species, in particular from the group of the gramineae still poses numerous problems at the present time.
Another disadvantage of these alternative transformation systems concerns as before, the relatively low transformation rates, which at this time have values of 1 to 5%.
These low transformation rates make it necessary to provide the DNA to be inserted with markers (for example antibiotics resistance genes), which make possible the rapid selection of the transformants from the large number of untransformed cells.
This means, however, that at the present time no satisfactory transformation process is available, which permits even the commercially efficient and cost efficient production of transgenic plants with novel and useful properties, in particular with regard to plants of the group of monocotyledoneae.
It is therefore an urgent task to develop processes making possible the rapid, efficient and reproducible transformation of all plants independently of their taxonomic position and the peculiarities resulting from it, thereby assuring the effective and economic production of transgenic plants, even commercially.
This is especially true relative to plants of the group of the monocotyledoneae, particularly those of the family of the gramineae, which includes the economically most important cultivated plants, such as wheat, barley, rye, oats, corn, rice, millet and the like, and which therefore are of a very special economic interest, particularly as no satisfactory process is available at this time for the preparation of transgenic monocotyledonous plants. Initial attempts in this direction consist of different, very recently developed transformation processes based on the insertion of DNA into plant cells included in a higher organized unit, such as for example an intact tissue body, an embryo or a whole, completely developed plant. This involves on the one hand the injection of exogenic DNA into the young inflorescence of rye plants (de la Pena & al., 1987), and on the other, a virus infection of corn plants, transmitted by Agrobacterium, with "maize streak virus" (Grimsley & al., 1987). However, these newly developed processes also have their disadvantages; thus for example, the process first mentioned above has not been reproducible to date.
An alternative process, again involving the transformation of plant cells within a higher organized unit, is based on the bombardment of said cells with particles associated with the DNA to be transformed. The impact of these highly accelerated particles produces holes in the cell walls of the cells impacted, through which the particles enter the cell, together with their associated DNA.
By means of these so-called microprojectiles a multitude of cells may be reached very rapidly. Microprojectiles have already been used in the past for gene transfer (Klein & al., 1988; Christou & al., 1988; EP-A-0 270 356) and were found to be suitable relative to certain problem definitions. However, the commercially available ballistic devices are not particularly suitable for the bombardment of small tissue areas with correspondingly small cells, such as for example in the case of the meristems. As the metal particles are fired dry in the ballistic processes, aggregates of a few or of many particles are fired which almost always tear deep wounds in the specimen. This tendency to aggregate is further enhanced by the bonding of the DNA on the particles.
The generally very high particle velocity of the ballistic processes requires a large working distance, which leads to the strong scattering of the particles. The particle velocity can be affected in the known ballistic methods in large stages only. It is hardly possible in this manner to improve the strong scattering of the particles. However, small tissues with small cells require dense coverage of the specimen with uniformly distributed individual particles in a narrowly limited target field, in order to obtain high transformation rates. The pulse of the particles should be as similar as possible, i.e. it should be possible to control the velocity and mass, the particle density on the specimen and the particle velocity very finely, so as to be able to adapt to different tissues.